Bottom Line:
The cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery.The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad.Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the beta-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli.

ABSTRACTThe cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery. Cif provokes cytopathic effects characterized by G(1) and G(2) cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21(waf1/cip1) and p27(kip1) and formation of actin stress fibres. The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad. Here we report the discovery and characterization of four Cif homologs encoded by different pathogenic or symbiotic bacteria isolated from vertebrates or invertebrates. Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the beta-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli. Although these Cif homologs are remarkably divergent in primary sequence, the catalytic triad is strictly conserved and was shown to be crucial for cell cycle arrest, cytoskeleton reorganization and CKIs accumulation. These results reveal that Cif proteins form a growing family of cyclomodulins in bacteria that interact with very distinct hosts including insects, nematodes and humans.

pone-0004855-g006: Transfection of CifYp induces cell cycle arrest in HeLa cells.HeLa cells were transfected with plasmid expressing GFP, GFP-CifEc or GFP-CifYp (wild-type (WT) or Cys variants (C/A)) fusion proteins. GFP expression and DNA content were analysed by flow cytometry 48 h post-transfection. Data are represented on two dimensional contour plot graphics with DNA content on the X-axis and GFP signal on the Y-axis. Gates corresponding to the GFP negative and GFP positive populations are indicated. Among the GFP positive populations, percentages of cells with 2N or 4N DNA content are indicated within the corresponding quadrants.

Mentions:
As it was not possible to introduce CifYp into cells using either the infection or BioPORTER treatments, the function of this protein was analysed directly by expressing cifYp in HeLa cells. CifYp and CifEc, used as a positive control, were expressed as a translational fusion with the fluorescent reporter protein GFP, allowing quantification of GFP-Cif expression in transfected cells. GFP alone was also transfected as a negative control. Among the GFP positive population, 96% of cells expressing GFP-CifEc had a 2N DNA content whereas the 2N population of cells expressing GFP alone was only 82% (Fig. 6). Consistent with previous studies demonstrating that Cif could also induce G1/S arrest [6], this result demonstrates that the cell cycle of transfected cells expressing GFP-CifEc was blocked in G1 (2N DNA content). As expected, the cell cycle arrest was not observed when the critical cysteine residue from the catalytic triad of CifEc was substituted (Fig. 6). Expression of GFP-CifYp in HeLa cells also led to accumulation of GFP-positive cells with 2N DNA content (96% against 82% for cells expressing GFP alone), demonstrating that CifYp induced a cell cycle arrest in G1 phase similarly to CifEc (Fig. 6). This result indicates that Cif from Y. pseudotuberculosis is a functional homolog of CifEc.

pone-0004855-g006: Transfection of CifYp induces cell cycle arrest in HeLa cells.HeLa cells were transfected with plasmid expressing GFP, GFP-CifEc or GFP-CifYp (wild-type (WT) or Cys variants (C/A)) fusion proteins. GFP expression and DNA content were analysed by flow cytometry 48 h post-transfection. Data are represented on two dimensional contour plot graphics with DNA content on the X-axis and GFP signal on the Y-axis. Gates corresponding to the GFP negative and GFP positive populations are indicated. Among the GFP positive populations, percentages of cells with 2N or 4N DNA content are indicated within the corresponding quadrants.

Mentions:
As it was not possible to introduce CifYp into cells using either the infection or BioPORTER treatments, the function of this protein was analysed directly by expressing cifYp in HeLa cells. CifYp and CifEc, used as a positive control, were expressed as a translational fusion with the fluorescent reporter protein GFP, allowing quantification of GFP-Cif expression in transfected cells. GFP alone was also transfected as a negative control. Among the GFP positive population, 96% of cells expressing GFP-CifEc had a 2N DNA content whereas the 2N population of cells expressing GFP alone was only 82% (Fig. 6). Consistent with previous studies demonstrating that Cif could also induce G1/S arrest [6], this result demonstrates that the cell cycle of transfected cells expressing GFP-CifEc was blocked in G1 (2N DNA content). As expected, the cell cycle arrest was not observed when the critical cysteine residue from the catalytic triad of CifEc was substituted (Fig. 6). Expression of GFP-CifYp in HeLa cells also led to accumulation of GFP-positive cells with 2N DNA content (96% against 82% for cells expressing GFP alone), demonstrating that CifYp induced a cell cycle arrest in G1 phase similarly to CifEc (Fig. 6). This result indicates that Cif from Y. pseudotuberculosis is a functional homolog of CifEc.

Bottom Line:
The cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery.The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad.Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the beta-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli.

ABSTRACTThe cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery. Cif provokes cytopathic effects characterized by G(1) and G(2) cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21(waf1/cip1) and p27(kip1) and formation of actin stress fibres. The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad. Here we report the discovery and characterization of four Cif homologs encoded by different pathogenic or symbiotic bacteria isolated from vertebrates or invertebrates. Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the beta-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli. Although these Cif homologs are remarkably divergent in primary sequence, the catalytic triad is strictly conserved and was shown to be crucial for cell cycle arrest, cytoskeleton reorganization and CKIs accumulation. These results reveal that Cif proteins form a growing family of cyclomodulins in bacteria that interact with very distinct hosts including insects, nematodes and humans.